Protein protease inhibitors appear to play a role in regulation of protease functions in living organisms and cells. Such protease inhibitors are widely distributed in animals and plants. Exemplary of these are alpha-1-antiprotease, soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor and antithrombin. Microbially produced protein protease inhibitors include a family of dimeric proteins, each 10 to 12 kilodaltons (kd). This family includes SSI, alkaline protease inhibitor (API-2) from S. griseoincarnatus, plasminostreptin (PSN) from S. antifibrinolyticus and a protease inhibitor from Streptoverticilium cinnamoneum. The inhibitors of the SSI family share extensive sequence homology, e.g., about 70% between SSI and PSN, but appear to have different protease specificities. See, generally, "Protein Protease Inhibitors--The Case of Streptomyces Subtilisin Inhibitor (SSI)", edited by Hiromi et al., Elsevier, 1985, pages 1-14, 139-161 and 365-395. Kakinuma et al., U.S. Pat. No. 4,014,860, disclose PSN and a producing strain thereof.
Protein protease inhibitors have medical application such as in treatment of lung tissue degradation caused by deficiency in alpha-1-antiprotease. Protein protease inhibitors can also be utilized to prevent protein degradation caused by proteases such as are present in serum. Wilson, EP-A-113,319, report use of Erythrina trypsin inhibitor to inhibit conversion of one chain tissue plasminogen activator to the two chain form which occurs in a presence of serum.
Streptomyces are an attractive host for production of desired polypeptides by recombinant DNA techniques because they possess the necessary cellular "machinery" to export proteins and because a great deal of experience in culturing Streptomyces for antibiotic production has been acquired.
A problem which has been encountered in production of heterologous proteins in Streptomyces is protein degradation by endogenous proteases. A second problem which has been encountered is in obtaining export signal sequences which can be fused to heterologous coding sequences to direct export or heterologous gene products.
Furthermore, it is desirable to obtain regulatory regions, e.g., promoters, ribosome binding sites and transcriptional enhancing/stabilizing sequences which can be used to express heterologous coding sequences in Streptomyces at high levels. Such promoters are typically associated with production of abundant mRNA and/or gene products.
Brawner et al., EP-A-187,630 disclose a Streptomyces beta-galactosidase gene expression unit and use of the promoter and of the export signal sequence thereof to express and export heterologous gene products.
It is an object of the present invention to provide novel protein protease inhibitors from Streptomyces. It is a further object to provide small exported proteins, which are exported and which are produced in abundant amounts, and DNA coding sequences, export signals and regulatory regions therefor.